The molecular events that mediate replication of hepatitis C virus (HCV) are largely obscure. The HCV nonstructural protein, NS5B, that synthesizes the viral positive strand RNA genome via (-) strand intermediate, is believed to be the key enzyme responsible for its replication but there are many unanswered questions about how its activity is controlled. Moreover, very few host cell protein(s)/factor(s) and other non-structural (NS) HCV proteins, which interact with NS5B and modulate its function, synthesis or turnover, have been identified and characterized. Proposed here, therefore, are two broad aspects, structure-activity relationship (SAR) optimization studies to design, and synthesize novel NS5B specific inhibitors and identification of cellular/viral protein(s) facilitating NS5B's activity upon interaction.
In Aim I, we will explore the potential of phenylalanine-derived non-nucleoside compounds in inhibiting NS5B's enzymatic activity. These studies will be guided by the resolved crystal structures of NS5B and its similarities with other polymerase's structures. NS5B contains a hydrophobic C-terminal tail that aids in membrane integration of the protein to form a replisome complex which is associated with cytoskeletal elements. However, the physical and functional interactions between NS5B and the cellular and other viral components of this complex are poorly understood.
In Aim II we propose to identify HCV nonstructural proteins interacting with NS5B using the ciphergen proteomic technology. For this, we will employ the UHCV-11 cell line, inducibly expressing the entire HCV open reading frame except NS5B. The kinetic parameters governing these interactions will be validated via surface plasmon resonance (Biacore).
In Aim III, we propose to identify host cell protein(s) from normal and HCV-infected liver explants, interacting with NS5B and thereby modulating its catalytic function. A critical understanding of NS5B, in perspective of its structure-function relationships and its combinatorial interaction with other proteins, will provide insights into the molecular effectors mediating HCV replication. This will enable us to understand, at the molecular level, the replication process of hepatitis C virus and will further facilitate the development of effective drugs/inhibitors against HCV.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK066837-02
Application #
6880099
Study Section
Special Emphasis Panel (ZRG1-GMA-2 (50))
Program Officer
Doo, Edward
Project Start
2004-04-01
Project End
2008-02-29
Budget Start
2005-03-01
Budget End
2006-02-28
Support Year
2
Fiscal Year
2005
Total Cost
$272,125
Indirect Cost
Name
University of Medicine & Dentistry of NJ
Department
Biochemistry
Type
Schools of Medicine
DUNS #
623946217
City
Newark
State
NJ
Country
United States
Zip Code
07107
Patel, Bhargav A; Krishnan, Ramalingam; Khadtare, Nikhil et al. (2013) Design and synthesis of L- and D-phenylalanine derived rhodanines with novel C5-arylidenes as inhibitors of HCV NS5B polymerase. Bioorg Med Chem 21:3262-71
Golub, Andriy G; Gurukumar, K R; Basu, Amartya et al. (2012) Discovery of new scaffolds for rational design of HCV NS5B polymerase inhibitors. Eur J Med Chem 58:258-64
Nichols, Daniel B; Fournet, Guy; Gurukumar, K R et al. (2012) Inhibition of hepatitis C virus NS5B polymerase by S-trityl-L-cysteine derivatives. Eur J Med Chem 49:191-9
Silberstein, Erica; Mihalik, Kathleen; Ulitzky, Laura et al. (2010) Persistent growth of a human plasma-derived hepatitis C virus genotype 1b isolate in cell culture. PLoS Pathog 6:e1000910
Talele, Tanaji T; Arora, Payal; Kulkarni, Shridhar S et al. (2010) Structure-based virtual screening, synthesis and SAR of novel inhibitors of hepatitis C virus NS5B polymerase. Bioorg Med Chem 18:4630-8
Rawal, Ravindra K; Katti, S B; Kaushik-Basu, Neerja et al. (2008) Non-nucleoside inhibitors of the hepatitis C virus NS5B RNA-dependant RNA polymerase: 2-aryl-3-heteroaryl-1,3-thiazolidin-4-one derivatives. Bioorg Med Chem Lett 18:6110-4
Kaushik-Basu, Neerja; Bopda-Waffo, Alain; Talele, Tanaji T et al. (2008) Identification and characterization of coumestans as novel HCV NS5B polymerase inhibitors. Nucleic Acids Res 36:1482-96
Kaushik-Basu, Neerja; Bopda-Waffo, Alain; Talele, Tanaji T et al. (2008) 4-Thiazolidinones: a novel class of hepatitis C virus NS5B polymerase inhibitors. Front Biosci 13:3857-68
Patel, Pallav D; Patel, Maulik R; Kaushik-Basu, Neerja et al. (2008) 3D QSAR and molecular docking studies of benzimidazole derivatives as hepatitis C virus NS5B polymerase inhibitors. J Chem Inf Model 48:42-55